Progressive morphologic changes in electrolytic brain lesions

EXPERIMENTAL

NEUROLOGY

Progressive

23,

529~536

(1969)

Morphologic Brain

Department

of Anatomy,

Mount New

Changes

in

Electrolytic

Lesions Sinai School York 10029

of Medicine,

New

York,

AND LEO Divisiorz

of Psychiatry,

Montefiore New

V. DICARA

Hospital, and Albert Einstein York, New York 10461

Received

Jnnzrary

College

of Medicine,

lo,1969

Lesions were produced in the caudate nucleus and in the hypothalamus of rats by passing anodal direct current through stereotaxically inserted electrodes. The rats were killed at various intervals from 1 hour to 16 weeks after operation and the changes in the microscopic appearance, configuration, and size of the lesions were studied. The main finding was that the apparent lesion size increases during the first postoperative day as cells fixed by the current disintegrate and then decreases progressively after the first postoperative week with contraction of the central cavity and disappearance of neuroglia cells. Introduction

In previous experiments on the behavioral effects of damage to various internal brain structures (2) we observed consistent changes in the size and shape of the lesions during postoperative recovery. The progressive histologic changes after cerebral insults of various types and in various structures have been studied in detail and are well known (5). Horsley and Clark (3) gave a detailed description of the microscopic appearance of electrolytic lesions of the cerebellum in animals killed immediately after and 3 weeks after operation. Also, Carpenter and Whittier (1) described distortions in the shapeof subcortical lesions as a function of postoperative survival. However, there has been no systematic parametric study of the changes in size, shape, and histologic appearance of internal electrolytic brain lesions as a function of postoperative recovery time. Since precise 1 Supported by USPHS grants MH 13189 and MH 16569 and done during first author’s tenure as an established investigator of the American Heart Association and second author’s tenure as an advanced research fellow of the American Heart Association. The authors are grateful to Dr. R. N. Eager for advice and assistance and to Drs. H. Weiner and S. Weisbroth for reading the manuscript. 529

530

WOLF

AND

DICARA

information on the nature of these changes is critical for correct interpretation of histologic data in lesion experiments, we undertook such a study. Method

Lesions were made in adult albino rats by passing a relatively constant anodal current through gauge-0 stainless steel insect pins insulated except for about 0.3 mm at the tip. The cathode was attached to the stereotaxic instrument. In each rat one lesion was placed in the right caudate nucleus and another in the left lateral hypothalamus. There were three groups of rats with lesions of one of three sizes in both placements: 0.8, 1.2, and 1.6 mamp. In all cases current was passed for 6 set and monitored on a milliammeter. Casesin which current was unstable during the 6 set were discarded. Rats were killed and perfused with formalin 1 hour, 1 day, 1, 2, 4, 8, and 16 weeks after lesion production. There were two rats for each lesion size and survival time-a total of 42 rats and 84 lesion samples. (However, in two casesa. and 1.2 mamp, 4 weeks-histologic preparations were not available, so that each of these points is represented by only one rat.) All brains were stored in 10% formalin for at least 1 month prior to sectioning to equalize any shrinkage which might occur as a result of fixation.

Sections

through

the lesions

were

cut on a freezing

microtome,

alternating one 100-p section to be stained by a modified Kliiver-Barrera method (6) and two 25-p sections to be stained with cresyl violet and with hematoxylin and eosin. respectively. The 100-p sections were used to determine lesion size and shape because with thick sections topographical relationships do not become distorted during the cutting and staining process. The volume of each lesion was determined hy drawing its outline from projected serial sections on graph paper and then computing volume by multiplying the total area enclosed by the projected outlines (‘determined by weighing the cut-out outlines) by a correction factor representing the amount of magnification and the distance between serial sections. The values arrived at by this procedure correlated almost perfectly with other measures,such as the cross-sectional area of the center of the lesion and the “lesion size index” of Carpenter and Whittier (1). The 25-p sections were used solely for cytological study. Results

Figure 1 shows photomicrographs of representative small (0.8 mamp) lesions in the caudate nucleus and in the hypothalamus for each of the seven survival times. The histopathologic changesare typical for all lesion sizesand regions. M~CUOSCOP~C .3ppeavancc.

MORPHOLOGIC

CHANGES

531

FIG. 1. Sections near centers of small (0.8 mamp) lesions in caudate nucleus (A) and hypothalamus(B) at each survival time. Samplespresentedin figures were chosenat randomfrom the two brains at eachpoint. Cresyl violet stain; magnification 15X.

One hour after making a lesion there is a central core of loose acellular necrotic debris, which may become detached during histologic preparation, leaving a cavity in the center of the lesion. The central core is surrounded by a narrow annulus containing a high concentration of cellular material apparently representing neuroglia cells, blood cells released by vascular disruption and remnants of lysed neurons. Immediately external to this dense rim is a narrow lightly stained zone containing very few cellular elements. Finally, there is a broad annulus of tissue containing cells which have been fixed by the current (coagulative necrosis), but which do not yet show

532

WOLF

AND

DICARA

clear signs of injury in cresyl violet (Fig. 1) or hematoxylin and eosin (Fig. 2A) stained material. This phenomenon is reminiscent of the “preservation” phenomenon described by Cajal, wherein one finds “necrosis associated with the most perfect morphological integrity” (5, p. 631). Finally, at 1 hour, there is no evidence of the commencement of inflammatory reaction.”

FIG. 2. Right halves of medium-sized (1.2 mamp) lesions in hypothalamus at 1 hour (A), 1 day (B), and 1 week (C) survival times. Hematoxylin and eosin stain ; magnification 100 X.

By 1 day after making a lesion, the inner dense rim of necrosis is still apparent, but much of the cellular debris has been removed and there is evidence that neutrophilic infiltration has commenced. The broad annulus of coagulated material which showed little evidence of damage at 1 hour, is now clearly abnormal. The neurons are in various stages of lysis and many of the cells have disintegrated completely, leaving the region relatively devoid of neurons in comparison to surrounding or contralateral tissue (Fig. 2B). At 1 week, the entire broad annulus region is filled with small cells presumably representing lymphocytes and neuroglia (Fig. 2C). At this point the healing process is clearly under way. At 2 weeks, a walling-off process becomes evident. (This can be seen especially clearly along the perimeter of the hypothalamic lesion in Fig. 1.) The reactive region is surrounded by a narrowed rim of dense gliosis and the entire region is contracting as cell remnants are removed. 2 The slow disintegration of the cells cannot be attributed to toxic metallic electrode deposits because the phenomenon has been observed when platinum clad electrodes which do not release metal ions are used (Mullan, S., M. Mailis, J. Karasi&, G. Vailati and F. Beckman. 1965. A reappraisal of the unipolar anodal electrolytic lesion. J. Nenrosurg. 22 : 531-,538.)

MORPHOIJOGIC

CHANGES

533

As can be seen in Fig. 1, from 2 weeks on there is a progressive diminution in the number of neuroglia cells, and the walled-off region continues to contract until there is only a narrow band of gliosis surrounding the central cavity. The cavity also contracts during this time. In the hypothalamus it often changes in shape from a smooth ovoid to an irregular stellate configuration, Lesion Size. Figure 3 shows lower magnification photomicrographs of sections through large lesions in the caudate nucleus and hypothalamus to demonstrate typical changes in lesion size and shape in relation to surrounding tissue. Figure 4 shows the mean volume of the lesions as a function of postoperative survival time for each of the three lesion sizes and each of the two regions. The “apparent lesion volume” represents the region of clearly abnormal tissue surrounding the tip of the electrode as described in detail above. Thus, it initially represents a region of cytolysis and acute inflammatory reaction and subsequently a region of neuroglial proliferation. It does not include the region of coagulative necrosis described in the l-hour samples since this tissue appears normal in standard histologic preparations. These criteria for determining lesion size presumably represent those most commonly employed by researchers in this field. As can be seen in the figures, the apparent lesion volume increases in size from 1 hour to 1 day ($ < .02 by t test of combined data). This increase is quite large, varying from 100 to 400% depending on the induced lesion size. The apparent lesion size attains a maximum between 1 day and 1 week and then diminishes progressively until the fourth week (p < .Ol), after which time the rate of contraction decreases abruptly so that only slight (and statistically insignificant) additional shrinkage occurs during the next 12 weeks. By 16 weeks after the induction of the lesion, its apparent volume has diminished to about 20% of its maximal extent. For a given structure and survival time, lesions produced by specified amounts and durations of anodal direct current were highly consistent in size and shape, thus confirming the findings of previous workers (4). However, identical amounts of current induce larger lesions in the hypothalamus than in the caudate nucleus with the hypothalamic lesions tending to be more ovoid in shape and to have a longer rostrocaudal axis. As can be seen in Fig. 4, this effect is highly consistent across current intensities and survival times and is significant (p < .Ol ). The reason for this difference is not clear. Possibly it may be related to a differential arrangement of myelinated fibers of high electrical resistance or to a differential sensitivity of the cells in the two regions to damage from electrical current. Finally, the degree of postoperative contraction of the lesions is greater in the hypothalamus than in the caudate nucleus (p < .05). This finding may

534

WOLF

AND

DICARA

MORPHOLO~

Post-lesion

535

CHANGES

survival

time

4. Mean apparent lesion volumes as a function of current intensity, lesion placement, and post-lesion survival time. C = caudate nucleus; H = hypothalamus. FIG.

be attributable simply to the fact that the hypothalamic lesions were generally larger than those in the caudate nucleus and thus the effect may have been due to differences in lesion size rather than lesion placement. However, it is possible that the proximity of the hypothalamic lesions to the third ventricle and the floor of the brain allows greater mobility of the surrounding tissue and thus more intense contraction of the lesion. Discussion

The major implication of the present study is that the apparent boundaries of electrolytic lesions do not invariably provide an accurate index of the actual amount of tissue damage. In specimens killed shortly after making lesions, as is common in acute electrophysiologic studies where lesions might be made to interrupt specific pathways, the extent of tissue damage may be grossly underestimated because necrotic cells are in a state of coagulative fixation and do not yet show signs of disintegration. In chronic

studies

in which

postoperative

survival

exceeds

1 week,

the de-

gree of underestimation of the tissue damage may increase concomitantly with progressive contraction of the lesion. In regions where the nuclei inFIG. 3. Sections near centers of large (1.6 mamp) lesions in caudate nucleus (A) and hypothalamus (B) at each survival time. Boundaries of lesions are outlined in black. Luxol blue and safranin-0 stain ; magnification 7 X .

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DICARA

ciuded in and surrounding the lesion are cytologically distinct and have clearly defined boundaries, it may be possible for a skilled worker to reconstruct accurately the initial extent of a contracted lesion. However, in a large, morphologically homogeneous structure or in regions of indistinct nuclear differentiation, precise reconstruction on the basis of morphologic criteria does not seem feasible (although partial reconstruction based on changes in cell density or other complex measures might be accomplished). In any case, common procedures for determining tissue damage are grossly inadequate for analysis of contracted lesions. For example, the common practice of tracing lesions from histologic sections on corresponding sections from a stereotaxic atlas can be especially misleading, not only in terms of estimating the over-all size of the lesion but also in localizing its placement, becausethe topographical relations of surrounding structures may be asymmetrically distorted concomitant with contraction of the lesion. In accordance with the present findings, it seemsthat for optimal precision and simplicity in anatomic delineation of brain damage from electrolytic lesions, the animal should be killed at a time when the apparent lesion volume is maximal. Under the present conditions, this time appears to be approximately between 1 day and 1 week, although the degree to which this time period is generalizable to other lesion methods and placements and to other speciesis, of course, uncertain. References M. B., and J. R. WHITTIER. 1952. Study of methods for producing experimental lesions of the central nervous system with special reference to stereotaxic technique. J. Cow@. Neurol. 97 : 73-117. DICARA, L. V., and G. WOLF. 1968. Bar pressing for food reinforcement after lesions of efferent pathways from lateral hypothalamus. Exptl. Neural. 21: 231-235. HORSLEY, V., and R. H. CLARK. 1908. The structure and functions of the cerebellum examined by a new method. Brain 31: 45-124. MACINTYRE, W. J., T. G. BIDDER, and V. ROWLAND. 1959. The production of brain lesions with electrical currents, pp. 723-732. 112 “Proceedings 1st National Biophysics Conference,” H. Quattler and H. J. Morowitz (Eds.). Yale University Press, New Haven. RAM~N Y CAJAL, S. 1928. “Degeneration and Regeneration of the Nervous System,” 2 vol. Oxford University Press, London. WOLF, G., and J. S. YEN. 1968. Improved staining of unembedded brain tissue. Physiol. Behavior 3 : 209-210.

1. CARPENTER,

2.

3. 4.

5. 6.